Project Summary Pseudomonas aeruginosa causes a variety of acute infections, such as ventilator-associated pneumonias, burn superinfections, neutropenic bacteremia, and medical device-related infections. In addition, P. aeruginosa chronically infects cystic fibrosis patients and causes significant morbidity and mortality in this population. With the increasing prevalence of antibiotic resistant organisms, developing agents that will modulate host immune responses will be key adjunctive treatments to conventional antibiotics in combating pathogens such as P. aeruginosa. In order to do this a detailed understanding of the innate immune pathways involved in recognition of pathogens such as P. aeruginosa are required. In this proposal we will examine the role of the NOD-like receptor (NLR) family member NLRC4 in recognition and response to infection with P. aeruginosa. In macrophages NLRC4 is activated in a multiprotein complex called the inflammasome upon infection with a wide variety of Gram-negative bacteria. The activation of the NLRC4 inflammasome ultimately results in the activation of the cysteine protease caspase-1 and its processing and secretion of proinflammatory cytokines. P. aeruginosa activates caspase-1 in an NLRC4-dependent manner leading to macrophage cell death and the release of interleukin (IL)-12 and IL-18. NLRC4-deficient mice also demonstrate increased susceptibility to infection with P. aeruginosa in vivo. In addition to Pseudomonas, other Gram-negative bacteria such as Salmonella, Legionella and Shigella have also been found to activate the NLRC4 inflammasome. One factor that is common to all these pathogens, and required for NLRC4 inflammasome activation, is a functional bacterial type III (T3SS) or type IV (T4SS) secretion system. However, it still remains unclear if NLRC4 serves as a cytosolic pattern recognition receptor for bacterial compounds that gain entry into the cytosol of the host cell through T3SS or T4SS, or if it indirectly senses the consequences of plasma membrane damage caused by the bacterial secretion systems. This proposal outlines three novel aims that will examine the molecular mechanism involved in activation of NLRC4: i) Defining the molecular components and cellular location for assembly of the NLRC4 inflammasome. ii) Elucidating the role of Toll-like receptors and reactive oxygen species in the upstream signaling pathways leading to NLRC4 inflammasome activation. iii) Determining if NLRC4 recognizes host plasma membrane components following their disruption by bacterial T3SS. PUBLIC HEALTH RELEVANCE: Project Narrative Pseudomonas aeruginosa causes a wide variety of opportunistic infections, particularly in hospitalized patient populations where it is responsibly for significant morbidity and mortality. P. aeruginosa is among the most common causes of ventilator associated pneumonia and amongst hospital acquired infections carries one of the highest mortality rates. With the increasing prevalence of multi-drug resistant strains on P. aeruginosa our treatment options are diminishing rapidly. Understanding how this pathogen interacts with and evades the immune system will help us identify new therapeutic targets to treat pseudomonal infections and is hence directly relevant to the VA patient care mission. Our studies will focus on how the innate immune pattern recognition receptor NLRC4 is activated by P. aeruginosa. In addition the information gained from the proposed studies will also shed light on the pathogenesis of related Gram-negative organisms.